This invention relates to novel tricyclic steroid analogs. More particularly, the invention relates to 1H-benz[e]indene dodecahydro compounds that are useful for enhancing gamma-aminobutyric acid (GABA)-induced chloride currents at the GABA receptor/chloride ionophore complex.
The importance of steroids as crucial components of cellular membranes is well established. Equally well established are the long term endocrine effects of steroid hormones. These endocrine effects are due to binding of steroids to intracellular receptors that subsequently interact with DNA and modulate gene expression. Less well delineated, but currently the subject of increasing interest, are the immediate modulatory effects of certain steroids at ion channels. For example, it is now established that steroid anesthetics, as well as certain endogenously produced metabolites of progesterone and deoxycorticosterone, facilitate GABA's ability to increase neuronal inhibition (for reviews see refs. 10, 15, 25). These steroid effects on GABAergic function have significant pharmacological, physiological, and pathological implications.
GABA is thought to be the major inhibitory neurotransmitter in the vertebrate central nervous system (CNS), exerting actions at two classes of receptors, designated GABA.sub.A and GABA.sub.B. These receptors can be distinguished physiologically and pharmacologically (5). GABA.sub.B sites represent the minority of CNS GABA receptors and, based on available studies, are unlikely to be a primary site of action for anesthetic/anticonvulsant drugs. In contrast, GABA.sub.A receptors appear to represent a major site of action for many CNS-active agents and are thought to be a site at which several classes of anesthetics exert their effects. At GABA.sub.A receptors, GABA promotes the direct opening of Cl.sup.- -selective ion channels. In most neurons, based on electrochemical gradients, the opening of these channels promotes the influx of Cl.sup.- and produces hyperpolarization of the neuronal membrane. A recurring theme in the GABA literature is that drugs which inhibit GABA.sub.A function, including the competitive antagonist bicuculline and the non-competitive antagonists picrotoxin, t-butylbicyclophosphorothionate (TBPS) and penicillin, act as convulsants whereas agents which augment GABA function, including benzodiazepines and barbiturates, act as anticonvulsants, anesthetics and sedatives (for review see ref. 49).
Consistent with Selye's (42) initial observations that steroids have CNS depressant properties, studies done over the past decade have provided evidence that the anesthetic actions of steroids may occur through enhancement of GABA.sub.A -mediated neuronal inhibition. Initial studies demonstrated that alphaxalone, an anesthetic steroid, prolongs the time course of GABA-mediated inhibitory synaptic responses in olfactory cortical slices (39). Subsequent studies using voltage clamp and single channel recording techniques have provided clear evidence that both anesthetic and endogenous steroids can alter GABA.sub.A receptor function in a variety of preparations. In cultured rat hippocampal and spinal cord neurons, anesthetic steroids augment Cl.sup.- currents produced by exogenous GABA (3, 24). Additionally, alphaxalone has been shown to open Cl.sup.- channels directly in the absence of GABA, at concentrations which are relevant to anesthetic effects (3). These steroid gated currents are blocked by bicuculline, suggesting that they are mediated through direct activation of GABA.sub.A receptors, perhaps by an action at the GABA recognition site. In addition to potentiating responses to exogenous GABA, alphaxalone augments inhibitory postsynaptic currents (IPSCs) mediated by GABA.sub.A receptors in cultured hippocampal neurons (14). This effect is manifest as a 5-8 fold prolongation of IPSC decay without change in peak IPSC amplitude or rise time. Taken together, these studies strongly suggest that both by direct Cl.sup.- channel activation and by modulation of GABA-mediated responses anesthetic steroids augment neuronal inhibition through modulation of the GABA.sub.A receptor complex.
The site at which steroids exert these effects remains unclear. Certain actions, including the direct gating of Cl.sup.- channels and the prolongation of IPSC decay are similar to the effects of anesthetic barbiturates (40). In addition, fluctuation analysis experiments have shown that alphaxalone, like the barbiturates, significantly prolongs the burst length of GABA-gated channels without changing the single channel conductance (3). However, recent studies using single channel recordings from recombinant human GABA.sub.A receptors expressed in human embryonic kidney cells have found differences in the actions of barbiturates and endogenous steroid metabolites. Whereas pentobarbital prolongs channel open times and burst lengths, 3.alpha.-OH-dihydroprogesterone (DHP) increases the frequency of channel opening without altering the open times (35). Other studies using ligand binding and Cl.sup.- flux measurements have demonstrated additive and synergistic effects of barbiturates and steroids, suggesting separate sites of action (11, 19, 47). Additionally, in bovine adrenal chromaffin cells, steroids greatly potentiate currents induced by high concentrations of pentobarbital (6). This suggests that either steroids and barbiturates act at separate sites in the GABA.sub.A complex or that the effects on GABA-gated responses are mediated by a site that is distinct from the site mediating direct Cl.sup.- channel gating.
An action of steroids at benzodiazepine receptors is less likely as several studies have failed to find an effect of the benzodiazepine antagonist R015-1788 (flumazenil) on steroid responses (6, 29). Additionally, benzodiazepine agonists potentiate GABA responses by increasing the apparent affinity of GABA for its receptor (7) without altering the single channel properties of the current (45). Benzodiazepine agonists also increase both the duration and amplitude of GABA-mediated IPSCs, an effect which differs from either the steroids or barbiturates (41). Finally, unlike steroids or barbiturates, benzodiazepines do not appear to gate Cl.sup.- channels directly in the absence of GABA.
Further complicating attempts to define the steroid site of action are observations that some steroid analogs inhibit GABA responses. Both pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) inhibit GABA currents in various CNS neurons (22, 23). However, PS, but not DHEAS, inhibits TBPS binding at the picrotoxin site, suggesting that these two agents may act at separate loci to affect GABA responses. Additionally, PS and picrotoxin have similar single channel effects, decreasing the opening frequency of the channels (27). These observations raise the possibility that the picrotoxin site may be responsible for some steroid actions. Previous studies using alkyl substituted .gamma.-butyrolactones have shown that both potentiation and inhibition of GABA.sub.A responses can be produced by agents acting at the picrotoxin site (16, 17). Interestingly, the 7-butyrolactones appear to alter GABA currents by changing the frequency of channel opening with less effect on the channel open times and no effect on the single channel conductance (2, 48).
Based on the data outlined above, it is clear that anesthetic and endogenous steroids can modulate GABA.sub.A receptor function. However there are several possibilities for the site(s) of action within the complex. The direct Cl.sup.- channel gating may be produced through an action at the GABA recognition site, based on the bicuculline sensitivity of the response. The alteration of responses produced by exogenous GABA and of IPSCs is more likely mediated through an allosteric site. Currently the putative barbiturate and picrotoxin sites are the leading candidates.
Steroid anesthetics were developed by the pharmaceutical industry decades before their effects at GABAergic neurons were established. The steroid anesthetic preparation, Althesin, was considered by anesthesiologists in Europe to have many of the properties desired for an intravenous anesthetic. These favorable properties have been discussed in an editorial by Morgan and Whitman (28). These authors also noted that Althesin was particularly effective and safe for use as an anesthetic in patients with high intracranial pressure resulting from severe head trauma. Unfortunately, Althesin is no longer available to anesthesiologists. It was removed from clinical use because of allergic reactions caused by the solubilizing agent used in the formulation.
Majewska has reviewed the role that steroid modulators of GABAergic function could play in the response to stress (25). Stress causes the release of CRF (corticotropin-releasing factor) from the hypothalamus. CRF in turn causes the release of adrenocorticotropic hormone (ACTH) from the pituitary, and ACTH then stimulates adrenal steroid biosynthesis. Among the adrenal steroids produced are cortisol and deoxycorticosterone. Cortisol has been shown to have biphasic actions at the GABA receptor channel complex found in guinea pig ileum (31). In picomolar concentrations it augments GABA-induced chloride currents, but at nanomolar concentrations it inhibits these same currents. Recent studies (36) support the hypothesis that the elevated concentrations of cortisol resulting from stress-induced increases in ACTH secretion could diminish neuronal inhibition by GABA and enhance the arousal state brought on by stress. Majewska further postulates that these effects may be enhanced by yet another endogenous steroid, pregnenolone sulfate, that she has shown to inhibit GABA-induced chloride currents (22). This sulfated steroid can be made not only in peripheral organs, but also by glial cells in the central nervous system (for a review of brain steroid biosynthesis see ref. 4). Finally, Majewska postulates that the deoxycorticosterone also released from the adrenal gland during stress has an important physiological function. Since deoxycorticosterone can be metabolized to THDOC (5.alpha.-pregnane-3.alpha., 21-diol-20-one), a steroid known to augment GABA-mediated neuronal inhibition (22), this steroid is postulated to counteract the effects of cortisol at the GABA receptor channel complex and restore homeostasis to the brain during stress.
This ability of endogenous steroids to either decrease or increase GABA-mediated neuronal inhibition has led many investigators (15, 20, 21, 25) to postulate that new steroid derivatives might be useful not only as anesthetics, but also as sedative hypnotics, anxiolytics, anticonvulsants, and antidepressants. In support of these potential uses for synthetic steroid derivatives that could modulate GABAergic function are the following results: 1) THDOC has shown both anxiolytic and sedative activity (different dose/repose curves) in two different animal models of anxiety (8); 2) THDOC has been shown to induce sleep and increase nonREM sleep in rats (26); 3) Saffan (a veterinary formulation of anesthetic steroids) at doses causing neurological symptoms, has been shown to have anticonvulsant activity against both maximal electroshock and chemically-induced seizures (33); and 4) depression is a frequent condition encountered in patients with Cushing's syndrome and it can be treated by lowering the elevated cortisol levels found in these patients (30).
The progesterone metabolite, 3.alpha.-OH-DHP (3.alpha.-hydroxy-5.alpha.-pregnan-20-one), shown below, is also thought to be an important physiologic regulator of GABA-mediated neuronal inhibition. This compound can be made de novo in brain or produced there from circulating progesterone (4). The observation that women having catamenial epilepsy, a condition in which seizure frequency changes during the menstrual cycle, have more seizures when progesterone levels are low during the menstrual cycle has led to the hypothesis that compounds mimicking the actions of 3.alpha.-OH-DHP may be useful as anticonvulsants and treatments for premenstrual syndrome (38). The later hypothesis is further supported by the fact that progesterone is often useful for treating premenstrual symdrome (25). ##STR2##
In summary, the effects of steroid modulation of GABA receptor channel function are highly significant. Steroid-induced hyperpolarization of GABAergic neurons is most likely the mechanism of action of anesthetic steroids. In addition, endogenously produced steroid metabolites of deoxycorticosterone and progesterone may be important physiological modulators of GABA-regulated neuronal inhibition.
Further background information on the structure/activity relationships in steroidal anesthetics can be had by reference to the review article by Phillips (34). Both of the above illustrated compounds, 3.alpha.-hydroxy-5.alpha.-pregnan-20-one and 3.alpha.-hydroxy-5.beta.-pregnan-20-one, are active in vivo as steroid anesthetics according to Phillips. These compounds also are potentiators of muscimol-stimulated chloride uptake in rat synaptoneurosomes (37) and potentiators of GABA-induced chloride currents in electrophysiological experiments (13, 32).